Facioscapulohumeral dystrophy (FSHD) is one of the most common inherited muscular dystrophies. The pathology is caused by a loss of epigenetic marks within the D4Z4 macrosatellite located in the sub-telomeric region of chromosome 4 leading to chromatin relaxation (1). In 95% of the FSHD patients (named FSHD1), this chromatin relaxation is associated with a contraction of the D4Z4 array (2). In the general population, this region is normally composed of 11 to 150 D4Z4 repeats, whereas FSHD1 patients only carry 1 to 10 repeats (3). The remaining 5% of the FSHD patients do not present a contraction of D4Z4 but 85% of them carry a mutation in the epigenetic modifier gene SMCHD1 (4). SMCHD1 is located on chromosome 18 and in most of the FSHD2 patients, the mutations lead to either a haploinsufficiency or a dominant negative mutations in SMCDH1 protein, leading to a reduced binding of SMCHD1 protein to the D4Z4 repeat and consequently to a loss of epigenetic marks in this region (4). In conclusion and despite the fact that 2 independent loci of the disease have been characterized, both FSHD1 and FSHD2 patients are undistinguishable and share a hypomethylation of D4Z4 on chromosome 4. This chromatin relaxation alone is not sufficient to trigger the disease and must be associated with a permissive chromosome 4 characterized by: (i) the presence of a permissive Stable Simple Sequence Length polymorphism (SSLP) located upstream D4Z4 (5-7). At least 12 different haplotypes have been characterized but only several are associated with FSHD (7, 8). These sequence variations may be important for the chromatin conformation but their exact roles in FSHD onset are unknown. (ii) the presence of a 4qA region containing a pLAM polyadenylation site distal to the last D4Z4 repeat allowing the stabilization of the DUX4 mRNA by the poly(A) tail (5, 9). Indeed, each D4Z4 repeat contains the open reading frame of a transcription factor named DUX4 (10, 11) and the chromatin relaxation results in an inefficient repression of this double homeobox gene in both FSHD1 and FSHD2. DUX4 is a transcription factor and DUX4-induced gene expression is the major molecular signature in FSHD skeletal muscles (12).
There is currently no effective treatment available for FSHD. A treatment of FSHD by preventing or inhibiting the expression of the DUX4 transcription factor has been proposed in application WO 2013/016352 using RNA interference based methods. However, direct gene inactivation methods using antisense technology or DNA-based gene deactivation through DNA enzyme cutting technologies (meganucleases, zinc finger nucleases, TALENs or others) may work well on FSHD patient cells but predictably will have low efficacy in vivo in the human. This is due to the fact that DUX4 gene transcription occurs haphazardly in a few myonuclei only at first. Subsequently neighbouring myonuclei are subject to the poison DUX4 protein effect modifying their gene expression (13). As a consequence of this poison peptide mechanism, whole organ- (and not cell-)treatment approaches will need to achieve a very high in tissue biodistribution in order to effectively inactivate DUX4 protein-transcribing myonuclei. This cannot be achieved at the present time where tissue biodistribution of OAN molecules or DNA cutting enzymes remains low (lit). In consequence, the method exposed herein targets the neutralization of the poison peptide DUX4 rather than the inactivation of the DUX4 gene.
In any case, no treatment is currently available for the FSHD patient. Therefore, an urgent need exists for providing a treatment of FSHD.